Functional stability of the second mitotic spindle in blastomeres and its association with multinucleation repair

During human embryonic development, early cleavage divisions are prone to errors, with abnormal division, non-segregation of chromosomes, and multinucleation often observed during the first mitosis. Some studies suggest that the nuclear status at the 2 cell stage does not affect blastocyst progression or implantation; however, chromosomal aneuploidy at the blastocyst stage is linked to the nuclear status at the 4-cell stage. We fluorescently labelled DNA and microtubules in 21 blastomeres of human 2-cell embryos and used confocal microscopy to study spindle morphology, chromosomal dynamics, and nuclear status during the second mitosis. We found significantly fewer multinucleated blastomeres in 4-cell embryos than in 2-cell embryos (22% vs. 56%: P=0.03). The second mitotic spindles were bipolar in 14 and multipolar in 7 of the 21 blastomeres. Most daughter cells from bipolar spindles were mononuclear. Further, the second mitotic bipolar spindles showed no defocusing poles. Pre-implantation genetic testing for aneuploidy analysis showed that multinucleation at the 4-cell stage did not necessarily reflect aneuploidy at the blastocyst stage. Errors in the first mitosis might be repaired in the second, suggesting that understanding early mitosis can improve embryo evaluation methods.